1. Field of the Invention
The present invention relates to a method for diagnosing a TSSE caused by a UTA strain, in a biological sample, by detecting PrP-res, and also to the use thereof in the context of differential diagnosis of the various UTA strains in a biological sample.
2. Description of Background Art
Transmissible spongiform subacute encephalopathies (TSSEs) are caused by unconventional transmissible agents (UTAs), also called prions, the precise nature of which to date remains disputed. TSSEs essentially comprise Creutzfeldt-Jakob disease in humans (CJD), scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE) in bovines. Other encephalopathies have been demonstrated in the Felidae, in mink or certain wild animals, such as deer or elk.
These diseases are always fatal and, at the current time, there is no effective treatment.
In TSSEs, there is an accumulation of a host's protein, PrP (or prion protein), in an abnormal form (PrP-res), mainly in the central nervous system; PrP-res copurified with infectiousness and accumulation thereof precedes the appearance of histological lesions. In vitro, it is toxic for cultures of neurons.
The two isoforms of PrP have the same amino acid sequence (see FIG. 1), but are different in their secondary structure: PrP-res has a significantly higher content of β-pleated sheets, whereas normal PrP (PrP-sen) has a greater number of α-helices.
Two biochemical properties make it possible, in general, to distinguish these two isoforms.
PrP-res is partially resistant to proteases, in particular to proteinase K (PK), which causes cleavage of its N-terminal end. After the action of PK, PrP-res is often called PrP27-30 because of the apparent molecular weight of the diglycosylated form; it is generally accepted that the cleavage site of PrP-res is located between amino acids 89 and 90 (Prusiner et al, Cell, 1984) for conventional strains.
PrP-res is insoluble in nonionic detergents, such as Triton X100 or Triton 114.
The normal form of the prion protein (PrP-sen) is, in principle, completely degraded by proteases and is entirely soluble in the presence of nonionic detergents.
The peptide sequences of hamster, human, bovine and ovine PrP-sen are presented in FIG. 1; they comprise, in particular, an octapeptide motif P(H/Q)GGG(-/T)WGQ (SEQ ID NO: 1), repeated 4 or 5 times, depending on the species. This octapeptide motif repeat corresponds to amino acids 51–91 of the PrP (numbering of the sequence of the human PrP) (B. Oesch et al., 1991).
For detecting the presence of the infectious agent, the most recent methods are based on selective detection of the abnormal PrP (PrP-res) linked to the infectious agent, by taking advantage of its partial resistance to proteases.
It is possible to distinguish:
Western blotting methods which are based on the immunological detection of PrP-res in a tissue extract, after treatment of the extract with a protease so as to destroy the normal isoform of the PrP (PrP-sen), separation of the proteins of the extract by electrophoresis, transfer onto a polymer membrane, and detection with a specific antibody which recognizes the PrP (Schaller O. et al., 1999),
tests of the ELISA type, which also involve treating tissue extract with a protease.
Among these various tests, which involve treating the tissue extracts with a protease, mention may be made of:
that described by Serban et al. (Neurology, 1990, 40, 110), who has developed a test for detecting PrP-res which includes immobilization of the proteins on a nitrocellulose membrane, followed by protease digestion, denaturation and immunodetection with monoclonal antibodies.
that described by Oesch et al. (Biochemistry, 1994, 33, 5926–5931), who have proposed, to quantify the amount of PrP-res, an immunofiltration assay for purifying PrP-res (ELIFA or enzyme-linked immunofiltration assay).
that described by Gratwohl et al., 1997, who propose an assay of the ELISA type. After treatment of the samples with proteinase K and purification of PrP-res by centrifugation, the latter is adsorbed onto microtitration plates and detected using rabbit polyclonal antibodies.
that described by Safar et al., 1998, who does not use proteinase K but compares the immunoreactivity of PrP-res immobilized on a solid support depending on whether or not it has been subjected to denaturing treatment.
In general, these various tests have the drawback of lacking sensitivity and thus of causing false-negatives.
Other methods propose treating the sample with denaturing products (Oesch et al., 1994 and 1999; WO 00/22438, The Reagents of the University of California), a limited treatment with proteinase K (WO 00/29850, Wallac Oy et al.) or treatment with a metallopeptidase (WO 00/22438), which make hidden antigenic sites accessible, which sites can be detected with the monoclonal antibody 3F4 which recognizes region 109–112 of PrP (WO 00/29850 or WO 00/22438).
The method described in WO 00/29850, Wallac Oy et al., has the major drawback of lacking specificity, due, in particular, to incomplete elimination of PrP-sen under the recommended treatment conditions, while the method described in WO 00/22438, The Regents of the University of California, lacks sensitivity due to the use of the detecting antibody 3F4 (WO 00/22438), which binds to only one motif on the protein.
The applicant has recently provided a test for the quantitative detection of PrP-res, which comprises a purification step which leads to significantly more sensitive detection and which represents a great advance for medical monitoring and assaying PrP-res in abattoirs. This method is, in particular, described in PCT international application WO 99/41280 and in a preliminary report of Directorate General XXIV of the European Commission (consumer policy and consumer health protection; http://europa.eu.int/comm/dg24/health/).
However, given the need for a particularly reliable test for diagnosing TSSEs, the applicant has continued its studies.